Mesenchymal stem cells are somatic stem cells, which have been found to exist in the bone marrow. Mesenchymal stem cells used as stem cells are capable of differentiating into bones, cartilage, and fats. Mesenchymal stem cells have been gaining attention as a potential cell source in cell therapy. Recently, it has been revealed that they also exist in the fetal appendage including the placenta, umbilical cord, and fetal membrane.
At present, mesenchymal stem cells have been gaining attention because of immunosuppressive capacity as well as differentiation capacity. There are ongoing clinical studies on hematopoietic stem cell transplantation for acute graft-versus-host disease (GVHD) and Crohn's disease which is an inflammatory bowel disease with the use of bone-marrow-derived mesenchymal stem cells. The present inventors conducted studies with an aim of realizing the applied use of fetal appendage-derived mesenchymal stem cells for immune-related diseases in clinical practice. Previously, the present inventors reported that fetal appendage-derived mesenchymal stem cells have differentiation capacity similar to that of bone marrow-derived mesenchymal stem cells (Non-Patent Document 6), fetal-membrane-derived mesenchymal stem cells can improve pathological conditions of rat autoimmune myocarditis models (Non-Patent Document 7), and umbilical cord-derived mesenchymal stem cells can improve the life-saving rate of mouse acute graft-versus-host disease (GVHD) models (Non-Patent Document 8). With the use of fetal appendage-derived mesenchymal stem cells, a large number of mesenchymal stem cells can be obtained at once, mass culture can be performed within a short period of time at low cost, non-invasive cell collection is possible, and high immunosuppressive effects can be obtained, compared with bone marrow-derived mesenchymal stem cells (Non-Patent Document 7). In consideration of the above, since MSCs such as fetal appendage-derived mesenchymal stem cells have remarkable immunosuppressive effects, they are applicable for cell therapy of various immune-related diseases.
Hitherto, a method for obtaining human fetal-derived pluripotent stem cells from the fetal appendage including the fetal membrane, placenta, and amniotic fluid has been reported (Patent Document 1). Patent Document 1 discloses a method for separating such stem cells which are regarded as c-kit (CD 117)-positive cells by flow cytometry. In addition, a method for obtaining stem cells/progenitor cells having capacity to differentiate into various adult and child cells from the placenta and umbilical cord has been reported (Patent Document 2). Patent Document 2 discloses a method for separating stem cells/progenitor cells having capacity to differentiate into cells that constitute various organs and tissues (=comparable or superior to differentiation capacity of mesenchymal stem cells) contained in the placenta and umbilical cord.
In general, degradative enzymes such as trypsin, collagenase and dispase have been used for separating cells including fetal-appendage-derived stem cells and progenitor cells (Patent Document 1 and 2 and Non-Patent Document 1 to 4). The fetal membrane included in the fetal appendage is divided into an amnion which is in contact with amniotic fluid and positioned closest to the fetus and a chorion which is positioned outside of the amnion. According to an ordinary method, fetal-membrane (amnion and chorion)-derived MSCs are also separated using degradative enzymes (Non-Patent Document 1 and 4).
The amnion which is a part of the fetal membrane of the fetal appendage is divided into the epithelial cell layer which is in contact with amniotic fluid and the extracellular matrix layer containing MSCs which is positioned under the epithelial cell layer (FIG. 1). Therefore, if the amnion as a whole is treated with trypsin, not only the extracellular matrix layer but also the basal membrane that supports the epithelial cell layer are digested, which results in a mixture of epithelial cells and MSCs. This has been problematic. In order to solve this problem, for example, Non-Patent Document 1 to 3 disclose methods for recovering MSCs, comprising removing epithelial cells in advance using a degradative enzyme or by a manual technique to separate high-purity MSCs from the amnion and treating the remaining extracellular matrix layer again with a separation enzyme. However, according to these methods, epithelial cells cannot be completely removed or the recovery amount of MSC decreases, which has been problematic.
Further, in order to realize the on-demand use of fetal membrane MSCs for cell therapy, cryopreservation is essential. At a research level, a variety of cells cryopreservation solutions containing dimethylsulfoxide (DMSO) as a base component have been commercially available. Also, a cryopreservation solution containing 10% DMSO has been used in clinical studies of bone marrow MSCs. However, DMSO-based cryopreservation solutions cause reduction of the cell survival rate after thawing, which has been problematic.